Dephosphorylation of human cyclin-dependent kinases by protein phosphatasetype 2C alpha and beta 2 isoforms

We previously reported that the activating phosphorylation on cyclin-depend ent kinases in yeast (Gdc28p) and in humans (Cdk2) is removed by type 2C pr otein phosphatases. In this study, we characterize this PP2C-like activity in HeLa cell extract and determine that it is due to PP2C beta2, a novel PP 2C beta isoform, and to PP2C alpha. PP2C alpha and PP2C beta2 co-purified w ith Mg2+-dependent Cdk2/Cdk6 phosphatase activity in DEAE-Sepharose, Superd ex-200, and Mono Q chromatographies. Moreover, purified recombinant PP2C al pha and PP2C beta2 proteins efficiently dephosphorylated monomeric Cdk2/Cdk 6 in vitro. The dephosphorylation of Cdk2 and Cdk6 by PP2C isoforms was inh ibited by the binding of cyclins. We found that the PP2C-like activity in H eLa cell extract, partially purified HeLa PP2C alpha and PP2C beta2 isoform s, and the recombinant PP2Cs exhibited a comparable substrate preference fo r a phosphothreonine containing substrate, consistent with the conservation of threonine residues at the site of activating phosphorylation in CDKs.